This invention spans the fields of nanotechnology and optical science. More particularly, it concerns the fields of nanolithography especially Dip-Pen Nanolithography (DPN) and the optical technique of surface enhanced Raman scattering (SERS). Dip-Pen Nanolithography (DPN), is a versatile technique in which a scanning probe microscope tip can be used to deliver a material (“ink”) to a surface via a water meniscus. DPN allows controlled deposition of suitable materials conventionally onto a flat substrate, with the size of the feature being written related to the complex interaction between the tip, ink, meniscus and surface. Further development of the technique has allowed Mirkin and co-workers to report direct and indirect writing of biological materials onto suitable surfaces to form highly structured arrays. DPN lithography can be carried out in both dot and line modes. Dot patterning involves the tip being moved vertically into contact by a Z piezoelectric motor action and the tip is lifted off between contact points. In line writing mode the tip staying in contact with the surface as it is moved by X and Y piezoelectric motors. Because the feature sizes produced by DPN are so small, detection of biological interactions is often achieved by atomic force microscopy (AFM: lateral force or tapping mode) or, as is most common in the case of DPN-directed DNA arrays, a fluorescence readout method is used.
Imaging and detection of single monolayers of a material or analyte at a flat surface is difficult. In the case of analysis by lateral force microscopy (LFM), imaging can only be effectively carried out where the surface is sufficiently flat to provide high contrast between areas containing the monolayer. Likewise in contact or tapping modes the imaging and detection quality is limited by the roughness of the underlying surface.
SERS is a highly sensitive spectroscopic technique that has been used in an increasing number of applications in biodiagnostics including gene probes, and DNA detection. The technique is flexible and with controlled chemistry can be performed using longer biologically suitable wavelengths of excitation (λex) in solution, or on nanostructured plasmonic gold surfaces. In surface enhanced Raman scattering the Raman signal from the analyte is enhanced dramatically by the proximity of the analyte to areas of high electric field. In SERS systems using nanoparticles or nanomaterials the field is generated on features of particular curvature or points where the oscillating electric field strength is strongest. A number of effective SERS substrates have been reported in recent years, including those fabricated by nanosphere lithography (NSL), silver metal island films and nanostructured gold surfaces. In SERS substrates the surface topography is always non-flat and the enhancement derives from areas of strong electric field gradient such as at sharp points (as in NSL) or standing waves set up in wells or cavities.
In addition to SERS, surface enhanced resonance Raman scattering (SERRS) potentially offers a number of significant advantages over other forms of detection such as fluorescence detection. When considering the simultaneous detection of multiple targets, it is significant to note that, in SERRS, a large proportion of the overall enhancement derives from the additional ‘resonance’ with the molecular chromophore. The resonance Raman spectrum from reporter dyes typically comprise fewer strong lines than would normally be expected from a larger molecule, as only certain vibronic states are probed using single wavelengths of excitation. This is a major advantage of the technique, when applied in a real assay, as a number of characteristic bands within each dye class are enhanced to a greater extent than other materials in the matrix. The characteristic narrow SERRS lines (˜0.5 nm width) have the potential to form the basis of an highly effective multiplexed analysis from a single excitation source. A further advantage of SERRS is that the excitation wavelength can be selected anywhere in the optical range and wavelength selectivity can be observed using some combinations of dye reporters.